Synapses made by a neuron with its synaptic partners are malleable during development, and as a consequence of experience, with respect to number, strength, and functional properties such as short and long term plasticity. A well studied model system for developmental, activity-dependent plasticity is mouse neuromuscular synapses, which undergo activity-dependent plasticity in development that is a hallmark of their smaller, less accessible counterparts in the CNS. During late embryonic and early postnatal life, neuromuscular synapses undergo synapse elimination, in which the synapses of one axon are pitted in competition against the synapses of other axons innervating the same target cell. Based on their activity patterns relative to their competitors, one or a small number of axons will emerge as winners, maintaining their synapses into adult life, while other axons lose the competition and are permanently deleted from neural circuitry. Despite many structural and a few functional studies of neuromuscular synapse elimination, little is known about the underlying mechanisms and many important questions remain to be addressed. One important set of questions includes how the structural changes in competing inputs are related to progressive changes in input strength, to the outcome of competition, and how activity mediates this process. The goal of this grant proposal is to understand the dynamics of the poorly understood presynaptic aspects of competition, including synaptic vesicle release, recycling and trafficking at developing mammalian neuromuscular synapses. We have developed transgenic lines of mice in which the Thy1 promoter drives expression of synaptopHluorin (Thy1-spH). SpH is a pH-sensitive variant of GFP tethered to the luminal domain of the vesicular protein VAMP2 that allows synaptic vesicle recycling to be monitored optically. Preliminary studies suggest that spH+ synaptic vesicle clusters can be readily visualized within motor axon terminals and vesicle release and trafficking assessed using optical measurements of activity- induced fluorescence changes in isolated sternomastoid nerve-muscle preparations as well as in vivo. Four aims are proposed, including (1) to determine the spatial and temporal dynamics of vesicle release across the terminal branches of developing and adult neuromuscular junctions;(2) to determine the relationship between vesicle release, synaptic strength and synaptic size of competing inputs to developing neuromuscular junctions undergoing synapse elimination;(3) to determine how postsynaptic activity blockade retrogradely affects synaptic vesicle release at developing and adult neuromuscular junctions;and (4) to determine how synaptic vesicles are trafficked among release sites within an individual terminal and how this is modulated by pre- and postsynaptic activity. Taken together, the aims proposed below will test the overall hypothesis that activity modulates presynaptic vesicle release and trafficking, affecting synaptic structure, strength and survival. This would provide a mechanism by which plastic changes in synaptic function could permanently alter neural circuitry. PUBLIC HEALTH RELEVANCE: I propose to study the mechanisms underlying synaptic competition during neural development, using neuromuscular synapses as a model system. Using transgenic mice in which the dynamics of synaptic vesicle recycling and trafficking can be monitored in vivo, I will test the overall hypothesis that activity modulates presynaptic vesicle release and trafficking, affecting synaptic structure, strength and survival. The results of the proposed experiments will provide fundamentally new insights into mechanisms by which activity changes synaptic function and neural circuitry during normal development, and contribute to understanding of developmental disorders such as epilepsy, autism and mental retardation, that have a significant public health impact.